A closer look into the microbiome of microalgal cultures.

Although bacteria are commonly co-occurring in microalgal cultivation and production systems, little is known about their community structure and how it might be affected by specific microalgal groups or growth conditions. A better understanding about the underlying factors that determine the growth of specific bacterial populations is not only important for optimizing microalgal production processes, but also in the context of product quality when the algal biomass is to be used for future food or feed. We analyzed the bacterial community composition associated with nine microalgal strains in stock culture, maintained in two different growth media, to explore how specific taxonomic microalgal groups, microalgal origin, or the growth medium affect the bacterial community composition. Furthermore, we monitored the bacterial community composition for three Phaeodactylum strains during batch cultivation in bubble columns to examine if the bacterial composition alters during cultivation. Our results reveal that different microalgal genera, kept at the same cultivation conditions over many years, displayed separate and unique bacterial communities, and that different strains of the same genus had very similar bacterial community compositions, despite originating from different habitats. However, when maintained in a different growth medium, the bacterial composition changed for some. During batch cultivation, the bacterial community structure remained relatively stable for each Phaeodactylum strain. This indicates that microalgae seem to impact the development of the associated bacterial communities and that different microalgal genera could create distinct conditions that select for dominance of specific bacteria. However, other factors such as the composition of growth medium also affect the formation of the bacterial community structure.

Protein Enrichment of Wheat Bread with Microalgae: Microchloropsis gaditana, Tetraselmis chui and Chlorella vulgaris.

Cell wall disrupted and dried Microchloropsis gaditana (Mg), Tetraselmis chui (Tc) and Chlorella vulgaris (Cv) microalgae biomasses, with or without ethanol pre-treatment, were added to wheat bread at a wheat flour substitution level of 12%, to enrich bread protein by 30%. Baking performance, protein quality and basic sensory properties were assessed. Compared to wheat, Mg, Tc and Cv contain higher amounts of essential amino acids and their incorporation markedly improved protein quality in the bread (DIAAS 57-66 vs. 46%). The incorporation of microalgae reduced dough strength and bread volume and increased crumb firmness. This was most pronounced for Cv and Tc but could be improved by ethanol treatment. Mg gave adequate dough strength, bread volume and crumb structure without ethanol treatment. To obtain bread of acceptable smell, appearance, and colour, ethanol treatment was necessary also for Mg as it markedly reduced the unpleasant smell and intense colour of all algae breads. Ethanol treatment reduced the relative content of lysine, but no other essential amino acids. However, it also had a negative impact on in vitro protein digestibility. Our results show that Mg had the largest potential for protein fortification of bread, but further work is needed to optimize pre-processing and assess consumer acceptance.

Predicting biomass and hydrocarbon productivities and colony size in continuous cultures of Botryococcus braunii showa.

Mathematical models were developed to predict biomass and hydrocarbon productivities and colony size (ouputs) of Botryococcus braunii showa cultures based on light intensity, temperature and dilution rate (inputs). These models predicted the following maximum values: biomass productivity, 1.3 g L-1 d-1; hydrocarbon productivity, 1.5 mg L-1 d-1; colony size, 320 µm under different culture conditions respectively. These values were confirmed experimentally. Additionally, the combination of inputs that simultaneously maximize all the possible outputs combinations were determined. The prediction for biomass-hydrocarbon-colony size were 1 g L-1 d-1, 12.05 mg L-1 d-1 and 156.8 µm respectively; biomass productivity-hydrocarbon productivities: 1 g L-1 d-1 and 13.94 mg L-1 d-1 respectively; biomass productivity-colony size: 1 g L-1 d-1 and 172.8 µm respectively; hydrocarbon productivity-colony size: 9 mg L-1 d-1 and 240 µm respectively. All these predictions were validated experimentally. These models might be very useful to implement a Botryococcus braunii showa large scale production.

Botryococcus braunii strains compared for biomass productivity, hydrocarbon and carbohydrate content.

Botryococcus braunii can produce both long-chain hydrocarbons as well as carbohydrates in large quantities, and is therefore a promising industrial organism for the production of biopolymer building blocks. Many studies describe the use of different strains of Botryococcus braunii but differences in handling and cultivation conditions make the comparison between strains difficult. In this study, 16 B. braunii strains obtained from six culture collections were compared for their biomass productivity and hydrocarbon and carbohydrate content. Biomass productivity was highest for AC768 strain with 1.8gL-1day-1, while hydrocarbon production ranged from none to up to 42% per gram biomass dry weight, with Showa showing the highest hydrocarbon content followed by AC761. The total carbohydrate content varied from 20% to 76% per gram of the biomass dry weight, with CCALA777 as the highest producer. Glucose and galactose are the main monosaccharides in most strains and fucose content reached 463mgL-1 in CCALA778.

Sorting cells of the microalga Chlorococcum littorale with increased triacylglycerol productivity.

BACKGROUND: Despite extensive research in the last decades, microalgae are still only economically feasible for high valued markets. Strain improvement is a strategy to increase productivities, hence reducing costs. In this work, we focus on microalgae selection: taking advantage of the natural biological variability of species to select variations based on desired characteristics. We focused on triacylglycerol (TAG), which have applications ranging from biodiesel to high-value omega-3 fatty-acids. Hence, we demonstrated a strategy to sort microalgae cells with increased TAG productivity. RESULTS: 1. We successfully identified sub-populations of cells with increased TAG productivity using Fluorescence assisted cell sorting (FACS). 2. We sequentially sorted cells after repeated cycles of N-starvation, resulting in five sorted populations (S1-S5). 3. The comparison between sorted and original populations showed that S5 had the highest TAG productivity [0.34 against 0.18 g l(-1) day(-1) (original), continuous light]. 4. Original and S5 were compared in lab-scale reactors under simulated summer conditions confirming the increased TAG productivity of S5 (0.4 against 0.2 g l(-1) day(-1)). Biomass composition analyses showed that S5 produced more biomass under N-starvation because of an increase only in TAG content and, flow cytometry showed that our selection removed cells with lower efficiency in producing TAGs. CONCLUSIONS: All combined, our results present a successful strategy to improve the TAG productivity of Chlorococcum littorale, without resourcing to genetic manipulation or random mutagenesis. Additionally, the improved TAG productivity of S5 was confirmed under simulated summer conditions, highlighting the industrial potential of S5 for microalgal TAG production.

Repeated nitrogen starvation doesn't affect lipid productivity of Chlorococcum littorale.

In the present work we wanted to know what happens during time to biomass and lipid productivities of Chlorococcum littorale repeatedly subjected to N-starvation. Experiments were done using repeated cycles of batch-wise N run-out (after 2days N=0). Two different cycles were used: repeated short-starvation (6days of N=0) over a total period of 72days and repeated long-starvation (13days of N=0) over a total period of 75days. Batches (using fresh inocula) were done separately as control. Shorter and longer periods of starvation showed no differences in biomass productivities and PSII quantum yield evolution. The repeated short-starvation-batches showed the same lipid productivities as the control short-starvation batches. Most importantly, the biomass lipid content was the same between control and repeated-batches. Altogether, the results point to C. littorale as a resilient and stable strain, with potential to be used under semi continuous cultivation.

Rapid method to screen and sort lipid accumulating microalgae.

The present work established an efficient staining method for fluorescence activated cell sorting (FACS) with Chlorococcum littorale maintaining cellular viability. The method was designed to detect high-lipid cells and to guarantee cellular viability. BODIPY505/515 (BP) was more suitable to FACS when compared to Nile red. The optimum concentrations were 0.4 µg ml(-1) of BP, 0.1% DMSO or 0.35% ethanol. Both ethanol and DMSO were equally efficient and assured cellular viability after the staining and sorting. Here a method is presented to rapidly screen and sort lipid rich cells of C. littorale with FACS, which can be used to produce new inoculum with increased cellular lipid content.

Analysis of fatty acid content and composition in microalgae.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

Phase toxicity of dodecane on the microalga Dunaliella salina.

In the so-called milking process of Dunaliella salina carotenoids are extracted and simultaneously produced by the culture, whilst the biomass concentration remains constant. Different theories exist about the extraction mechanisms although none have been proven yet. In this research, direct contact between dodecane and cells during the extraction process was studied microscopically and effects of direct contact were determined during in situ extraction experiments. Our results showed that water-solvent interphase contact resulted in cell death. This cell death and consequent cell rupture resulted in the release and concomitant extraction of the carotenoids. Furthermore, it has been suggested to add a small amount of dichloromethane to the biocompatible dodecane to create an organic phase with more extraction capacity. Our results showed that the addition of dichloromethane resulted in increased cell death and consequently the extraction rate increased. The improved solubility of carotenoids in an organic phase with dichloromethane did not significantly increase the extraction rate.

Continuous production of carotenoids from Dunaliella salina.

During the in situ extraction of ß-carotene from Dunaliella salina, the causal relationship between carotenoid extraction and cell death indicated that cell growth and cell death should be at equilibrium for a continuous in situ extraction process. In a flat-panel photobioreactor that was operated as a turbidostat cell numbers of stressed cells were kept constant while attaining a continuous well-defined light-stress. In this way it was possible to study the balance between cell growth and cell death and determine whether both could be increased to reach higher volumetric productivities of carotenoids. In the two-phase system a volumetric productivity of 8.3 mg ß-carotene L(RV)(-1)d(-1) was obtained. In situ extraction contributed only partly to this productivity. The major part came from net production of carotenoid-rich biomass, due to a high growth rate of the cells and subsequent dilution of the reactor. To reach equilibrium between cell growth and cell death, sparging rates of dodecane could have been increased. However, already at the applied sparging rate of 286 L(dod)L(RV)(-1)min(-1) emulsion formation of the dodecane in the aqueous phase appeared. In a turbidostat without in situ extraction a volumetric productivity of 13.5 mg ß-caroteneL(RV)(-1)d(-1) was reached, solely based on the continuous production of carotenoid-rich biomass.

Two-phase systems: potential for in situ extraction of microalgal products.

Algae are currently used for production of niche products and are becoming increasingly interesting for the production of bulk commodities, such as biodiesel. For the production of these goods to become economically feasible, production costs will have to be lowered by one order of magnitude. The application of two-phase systems could be used to lower production costs. These systems circumvent the costly step of cell harvesting, whilst the product is extracted and prepared for downstream processing. The mechanism of extraction is a fundamental aspect of the practical question whether two-phase systems can be applied for in situ extraction, viz, simultaneous growth, product formation and extraction, or as a separate downstream processing step. Three possible mechanisms are discussed; 1) product excretion 2) cell permeabilization, and 3) cell death. It was shown that in the case of product excretion, the application of two-phase systems for in situ extraction can be very valuable. With permeabilization and cell death, in situ extraction is not ideal, but the application of two-phase systems as downstream extraction steps can be part of a well-designed biorefinery process. In this way, processing costs can be decreased while the product is mildly and selectively extracted. Thus far none of the algal strains used in two-phase systems have been shown to excrete their product; the output has always been the result of cell death. Two-phase systems can be a good approach as a downstream processing step for these species. For future applications of two-phase in situ extraction in algal production processes, either new species that show product excretion should be discovered, or existing species should be modified to induce product excretion.

Carotenoid fluorescence in Dunaliella salina.

Dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity. The produced carotenoids are mainly stored in lipid globules. For various research purposes, such as production and extraction kinetics, we would like to determine and/or localise the carotenoid globules in vivo. In this study, we show that the carotenoid-rich globules emit clear green fluorescence, which can be used in, for example, fluorescence microscopy (e.g. CLSM) to obtain pictures of the cells and their carotenoid content.

The selectivity of milking of Dunaliella salina.

The process of the simultaneous production and extraction of carotenoids, milking, of Dunaliella salina was studied. We would like to know the selectivity of this process. Could all the carotenoids produced be extracted? And would it be possible to vary the profile of the produced carotenoids and, consequently, influence the type of carotenoids extracted? By using three different D. salina strains and three different stress conditions, we varied the profiles of the carotenoids produced. Between Dunaliella bardawil and D. salina 19/18, no remarkable differences were seen in the extraction profiles, although D. salina 19/18 seemed to be better extractable. D. salina 19/25 was not "milkable" at all. The milking process could only be called selective for secondary carotenoids in case gentle mixing was used. In aerated flat-panel photobioreactors, extraction was much better, but selectiveness decreased and also chlorophyll and primary carotenoids were extracted. This was possibly related to cell damage due to shear stress.